Corticosteroid treatment is associated with increased filamentous fungal burden in allergic fungal disease

Background Allergic diseases caused by fungi are common. The best understood conditions are allergic bronchopulmonaryaspergillosis (ABPA) and severe asthma with fungal sensitisation (SAFS). Our knowledge of the fungal microbiome (mycobiome) is limited to a few studies involving healthy individuals, asthmatics and smokers. No study has yet examined the mycobiome in fungal lung disease. Objectives The main aim of this study was to determine the mycobiome in lungs of individuals with well characterised fungal disease. A secondary objective was to determine possible effects of treatment on the mycobiome. Methods After bronchoscopy, ITS1 DNA was amplified and sequenced and fungal load determined by RT-PCR. Clinical and treatment variables were correlated with the main species identified. ABPA (n=16), SAFS (n=16), severe asthma not sensitised to fungi, (n=9), mild asthma patients(n=7) and 10 healthy controls were studied. Results The mycobiome was highly varied with severe asthmatics carrying higher loads of fungus. Healthy individuals had low fungal loads, mostly poorly characterised Malasezziales.The most common fungus in asthmatics was Aspergillus fumigatus complex and this taxon accounted for the increased burden of fungus in the high level samples. Corticosteroid treatment was significantly associated with increased fungal load (p<0.01). Conclusions The mycobiome is highly variable. Highest loads of fungus are observed in severe asthmatics and the most common fungus is Aspergillus fumigatus complex. Individuals receiving steroid therapy had significantly higher levels of Aspergillus and total fungus in their BAL

Identification of a Putative Crf Splice Variant and Generation of Recombinant Antibodies for the Specific Detection of Aspergillus fumigatus

BACKGROUND: Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA) in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of recombinant antibodies for the detection of A. fumigatus by ELISA. RESULTS: The A. fumigatus antigen Crf2 was isolated from a human patient with proven IA. It is a novel variant of a group of surface proteins (Crf1, Asp f9, Asp f16) which belong to the glycosylhydrolase family. Single chain fragment variables (scFvs) were obtained by phage display from a human naive antibody gene library and an immune antibody gene library generated from a macaque immunized with recombinant Crf2. Two different selection strategies were performed and shown to influence the selection of scFvs recognizing the Crf2 antigen in its native conformation. Using these antibodies, Crf2 was localized in growing hyphae of A. fumigatus but not in spores. In addition, the antibodies allowed differentiation between A. fumigatus and related Aspergillus species or Candida albicans by immunofluorescence microscopy. The scFv antibody clones were further characterized for their affinity, the nature of their epitope, their serum stability and their detection limit of Crf2 in human serum. CONCLUSION: Crf2 and the corresponding recombinant antibodies offer a novel approach for the early diagnostics of IA caused by A. fumigatus

Clinical performance of FXG: RESP (Asp+) assay for Pneumocystis jirovecii on respiratory specimens

Objectives: Pneumocystis pneumonia continues to be a common infection in HIV patients and also occurs in other immunocompromised patients. Early diagnosis is known to improve outcome, and specifically, exclusion of the diagnosis, reduces the need for toxic empirical high dose cotrimoxazole therapy. Real-Time PCR offers the prospect of faster and highly sensitive detection of P. jirovecii. FXG : RESP (Asp +) [Myconostica, UK] is a test kit that detects both P. jirovecii and Aspergillus spp., utilising molecular beacons. In this report we focus on the clinical performance for P. jirovecii. Methods: The FXG : RESP (Asp +) real-time PCR kit utilises the large subunit mtRNA gene as a target to detect P. jirovecii and the assay is highly sensitive, being able to detect 6 target copies. The assay appears to be specific for Pneumocystis spp, with the possible exception of Fusarium solani. It was tested blindly on 196 BAL samples, collected from 4 European hospitals. All results were compared to microscopy, usually Calcofluor or Gomori methanamine silver stains, performed shortly after the sample was collected, and in non-AIDS patients whether patient was treated for PCP. Most HIV/AIDS samples had been previously extracted and stored frozen as DNA; the remainder were extracted with the MycXtra™ fungal DNA extraction kit, having been stored frozen unprocessed. Results: The kit contains an internal amplification control to detect potential inhibition of the PCR reaction and 6 (3%) of the clinical samples showed evidence of inhibition. These results were excluded from analysis, although 5 were positive by both microscopy and the FXG : RESP (Asp +) assay, and would be reported clinically. 42 samples were from HIV/AIDS patients and 148 from other patients, mostly with leukaemia. With respect to Pneumocystis detection, the FXG : RESP (Asp +) assay had good performance with a sensitivity of 97.4%, specificity of 92.9%, positive and negative predictive values of 90.4% and 98.1%. 8 samples had negative microscopy but very high FXG assay signals, suggesting that the microscopy was falsely negative, as reported in prior literature. These were reported as false positives Conclusions: The clinical performance of the FXG : RESP (Asp +) assay for the diagnosis of Pneumocystis pneumonia is superb. Overall the speed of detection and sensitivity of the FXG : RESP (Asp +) assay should bring substantial clinical benefits. Prospective and supportive clinical trials are ongoing.status: publishe

Emergence of azole-resistant Aspergillus fumigatus strains due to agricultural azole use creates an increasing threat to human health.

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